Figure 5.

The TAAR1 small-molecule agonist RO5256390 induces pCREB, Irs-2, pERK, calcium signaling, proliferation, and GSIS in Ins-1 cells. A and C, Western blots of CREB phosphorylation (A) or ERK1/2 phosphorylation (C) in Ins-1 cells 10 min after RO5256390 (0–10 μm) or forskolin (0.3 μm). Representative blots of pCREB or pERK1/2 from one of at least three independent experiments are shown, and blots were stripped and reprobed with either β-actin or total ERK1/2 as a loading control. B, quantitative PCR of Irs-2 gene expression (1 h) induced by RO5256390 (10 μm). Data are expressed as mean ΔΔCT (± S.D.) of Irs-2, using Gapdh as the housekeeping gene, and were analyzed by Student's t test (n = 3). ****, p < 0.0001. D, RO5256390 induces calcium flux in Ins-1 cells (1.5 mm extracellular calcium). E, RO5256390 (10 μm) and forskolin (0.3 μm) increase [³H]thymidine incorporation into Ins-1 cells (24 h). F, insulin secretion in response to TAAR1 agonists (RO5256390 and T₁AM) or modulators of TAAR1/cAMP-dependent signaling were added to cells at either 3.5 mm (low) or 20 mm (high) glucose. Insulin secretion (2 h) was determined by ELISA (mean ± S.D.) and analyzed by one-way ANOVA (global p values are shown) using Dunnett's multiple comparison post hoc test (E, n = 6) or Newman-Keuls test (F, n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.